1. Field of the Invention
Fluorescent compounds find wide application because of their ability to emit light upon excitation with energy within certain energy ranges. By virtue of this ability, fluorescers have found employment as labels in chemical or biological processes, e.g. assays. That is, various compounds can be conjugated to a fluorescent compound, the conjugate subjected to some type of partitioning, and the fate of the conjugate determined by irradiating the sample with light and detecting the zone in which the conjugate exists.
This technique can be employed in immunoassays, involving specific binding pairs, such as ligands and receptors, e.g., antigens and antibodies. By conjugating a fluorescer to one of the members of the specific binding pair and employing various protocols, one can provide for partitioning of the fluorescer conjugate between a solid phase and a liquid phase in relation to the amount of antigen in an unknown sample. By measuring the fluorescence of either of the phases, one can then relate the level of fluorescence observed to a concentration of the antigen in the sample.
Alternatively, one can avoid partitioning of the fluorescent label by providing for a mechanism which varies the fluorescence of the label, depending upon the label environment in a liquid medium. For example, in addition to labeling one of the members of the specific binding pair with the fluorescer, one may label the other member with a quencher, that is, a molecule which is able to absorb the excitation energy of the fluorescer molecule, preventing the emission of a photon. The quenching then will occur only when the two members of the specific binding pair are associated and the fluorescer and quencher thereby achieve the required spatial proximity for quenching.
2. Description of the Prior Art
U.S. Pat. No. 3,998,943 discloses an immunoassay involving a ligand-fluorescer conjugate employing steric inhibition of simultaneous binding of antibody for ligand and antibody for fluorescer, where the antibody for fluorescer substantially quenches the fluorescence. U.S. Pat. No. 3,996,345 describes an immunoassay involving fluorescer-quencher pairs, which employs a conjugate of a fluorescer bonded to one member of a specific binding pair and a conjugate of a quencher bonded to the same or different member of a specific binding pair. The assay is dependent upon the degree to which the quencher and fluorescer are brought within quenching proximity based on the amount of analyte in the medium. Novel conjugates of fluorescers and quenchers with poly(amino) acids are disclosed in U.S. Pat. Nos. 4,351,760 and 4,318,846. A dye tagged reagent is described in U.S. Pat. No. 4,166,105. Digoxigenin immunogens, antibodies, labeled conjugates and related derivates are discussed in U.S. Pat. No. 4,469,797.
Various squarate dyes are discussed by Sprenger, et al., Angew. Chem., 80, 541 (1968) [Angew. Chem. internatl Edit, Vol. 7: 530-535 1968]; Sprenger, et al., Angew. Chem., 79; 581, 1967; Sprenger, et al., Angew. Chem. internat. Edit., 5: 894, 1966; and Maahs, et al., ibid., 5: 888, 1966.
The use of laser beams and slits to differentiate particles based on their relative size by the correlation of fluorescence fluctuations in a relatively large sample volume is described by Briggs et al., Science, 212: 1266-1267, 1981, and by Nicoli et al., Proc. Natl. Acad. Sci., USA, 77: 4904-4908, 1980.